The efficacy of DNA MMR enzyme immunohistochemistry as a ... · [5], only 15 had detectable...

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The efficacy of DNA MMR enzyme immunohistochemistry as a screening test for

hypermutated gliomas

Matthew McCord, MD,1 Alicia Steffens,2 Rodrigo Javier,2 Kwok-Ling Kam, MBBS,1 Kathleen

McCortney, MS,2 and Craig Horbinski, MD PhD1, 2

1Department of Pathology, Northwestern University, Chicago, IL 60630, USA

2Department of Neurosurgery, Northwestern University, Chicago, IL 60630, USA

CORRESPONDING AUTHOR

Craig Horbinski, M.D., Ph.D.

Northwestern University

Tarry 2-705

300 East Superior St.

Chicago, IL, 60611

Phone: 312-503-6127

Email: [email protected]

All rights reserved. No reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (whichthis version posted November 15, 2019. ; https://doi.org/10.1101/19012005doi: medRxiv preprint

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

https://doi.org/10.1101/19012005

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ABSTRACT

A subset of gliomas has DNA repair defects that lead to hypermutated genomes. While such

tumors are resistant to alkylating chemotherapies, they may also express more mutant

neoantigens on their cell surfaces, and thus be more responsive to immunotherapies. A fast,

inexpensive method of screening for hypermutated gliomas would therefore be of great clinical

value. Since immunohistochemistry (IHC) for the DNA mismatch repair (MMR) proteins Msh2,

Msh6, Mlh1, and Pms2 is already used to screen for hypermutated colorectal cancers, we

sought to determine whether that panel might have similar utility in gliomas. MMR IHC was

scored in 100 WHO grade I-IV gliomas with known tumor mutation burdens (TMB), while

blinded to TMB data. Eight of 100 cases showed loss of one or more MMR proteins by IHC, and

all 8 were hypermutated. Among the remaining 94 gliomas with intact MMR IHC, only one was

hypermutated; that tumor had an inactivating mutation in another DNA repair gene, ATM.

Overall accuracy, sensitivity, and specificity were 99%, 89%, and 100%, respectively. The

strongest correlates with hypermutation were prior TMZ treatment, MGMT promoter methylation,

and IDH1 mutation. Among MMR-deficient hypermutated gliomas, 50% contained both MMR-

lost and MMR-retained tumor cells. Together, these data suggest that MMR IHC could be a

viable front-line screening test for gliomas in which immunotherapy is being considered. They

also suggest that not all cells in a hypermutated glioma may actually be MMR-deficient, a

finding that might need to be considered when treating such tumors with immunotherapies.

KEY WORDS

Glioma, hypermutator, MGMT, IDH1, temozolomide, mismatch repair



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INTRODUCTION

Gliomas are the most common primary brain tumors in adults [19]. Standard of care

includes surgical resection followed by adjuvant radiation and temozolomide (TMZ), a DNA

alkylating agent [24]. However, tumors nearly always recur and lose sensitivity to adjuvant

therapy.

When GBMs are challenged with TMZ, recurrent subclones often emerge with

inactivating mutations in genes encoding DNA mismatch repair (MMR) enzymes, most

commonly MSH2, MSH6, MLH1, and PMS2 [7, 11, 28]. Glioma tumor mutation burden (TMB) is

normally ~1 mutation per megabase (Mb) of DNA [2], but MMR defects can lead to a

“hypermutator” phenotype, defined as TMB >20 mutations/Mb DNA [10, 11, 14]. Hypermutated

tumor cells tend to display more mutant proteins on their surfaces, making them potentially

more vulnerable to immunotherapies like PD-1 and PD-L1 checkpoint inhibitors [4, 9]. Other

forms of hypermutated cancers have shown promising responses to immune checkpoint

inhibitors [20], and there is great interest in this strategy for hypermutated gliomas [6, 10].

Next generation sequencing (NGS) is the current gold standard for detecting DNA MMR

defects and quantifying TMB, but larger panels are costly and have prolonged turnaround time.

Targeted NGS panels, such as Glioseq [18], are less expensive and faster, but usually do not

screen for MMR mutations and do not cover enough of the genome to reliably determine TMB.

A standardized quartet of IHC stains (Msh6, Msh2, Mlh1, and Pms2) is used to detect

loss of normal MMR gene expression in colorectal cancers [22]. Because most pathology

laboratories already have this MMR IHC panel for routine use, we sought to determine its utility

as a screening test for hypermutated gliomas.



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METHODS

The cohort consisted of 100 World Health Organization (WHO) grade I-IV gliomas from

the Northwestern Nervous System Tumor Bank with known TMB and MMR gene mutations, as

determined by Tempus xT NGS covering approximately 600 genes (Table 1). Case collection

was done under a Northwestern Institutional Review Board-approved protocol. IHC was

performed using 4 different primary antibodies, including Msh2 (Cell Marque G219-1129,

1:700), Msh6 (Cell Marque 44 (287M-15), 1:100), Mlh1 (Leica NCL-L-MLH1, 1:100), and Pms2

(Cell Marque MRQ-28 (288M-15), 1:50). Formalin-fixed, paraffin-embedded 4 µm thick tissue

sections were baked at 60°C for 30-60 minutes before being deparaffinized and re-hydrated.

Antigen retrieval for Msh6, Msh2, and Pms2 was achieved using a Universal Retrieval (Abcam)

buffer in a decloaking chamber reaching 110°C for 5-20 minutes. Antigen retrieval for Mlh1 used

a citrate buffer (pH 6) in a decloaking chamber reaching 110°C for 10 minutes. Slides were

cooled to room temperature and washed in TBS before neutralizing endogenous peroxidase

(Biocare Peroxidase 1). Slides were then treated with a serum-free casein background block

(Biocare Background Sniper) before pre-incubation in a 10% goat serum block for 60 minutes.

Primary antibody was added to the slides for overnight incubation at 4°C. After incubation,

slides were washed in 3 5-minute washes with TBS-T before incubating in HRP polymer

(Biocare MACH 4 Universal HRP Polymer). Reaction products were visualized with DAB

(Biocare Betazoid DAB Chromogen Kit). Slides were counterstained with hematoxylin,

dehydrated, and mounted with xylene-based mounting media.

Each IHC marker was examined under light microscopy by two independent reviewers

(MM and CH) while blinded to NGS data and TMB. Each IHC marker was scored as “retained”

or “lost.” In cases with lost MMR expression, the pattern (homogeneous versus heterogeneous)

was noted. Nonneoplastic cells within each glioma (e.g. lymphocytes, endothelial cells) served

as internal positive controls. Interobserver discrepancies were resolved by reviewing equivocal

cases together. Regression analyses were performed using http://vassarstats.net/multU.html



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RESULTS

The cohort of 100 gliomas with NGS and TMB data included 70 grade IV GBMs, 13

grade III astrocytomas, 4 grade II astrocytomas, 2 grade III oligodendrogliomas, 7 grade II

oligodendrogliomas, and 4 grade I gliomas (Table 1). Eight (8%) showed loss of at least one

MMR protein by IHC (Figure 1, Table 2). All 8 gliomas with MMR loss had TMB >20, and all 8

had mutations and/or copy number losses in corresponding MMR genes (Table 2). Of the

remaining 92 gliomas with intact MMR IHC, only one was hypermutated (TMB=29.5). That

glioma did not have mutations in MSH2, MSH6, MLH1, or PMS2, but instead contained an

inactivating splice site mutation in ATM (Table 2). Sensitivity, specificity, and overall accuracy of

MMR IHC for identifying hypermutated gliomas in this cohort was 88%, 100%, and 99%,

respectively.

All 9 hypermutated gliomas had MGMT promoter methylation and were post-TMZ

recurrences, and 5/9 (56%) were IDH1 mutant (Table 2). Correlation and multiple regression

analyses confirmed that the variables most strongly associated with hypermutation were prior

treatment with TMZ, MGMT methylation, and IDH1 mutation (Table 3 and Table 4).

In 4 of 8 gliomas with lost MMR expression, the pattern of loss was clearly

heterogeneous, as some tumor cells retained all MMR enzymes, while other cells lost

expression of one or more MMR enzymes (Figure 2).



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DISCUSSION

Despite their generally aggressive behavior, gliomas tend to have low TMB relative to

most other kinds of cancer [2]. However, gliomas that are hypermutated, either at initial

presentation or recurrence, may be ideal targets for immunotherapy. Such gliomas usually show

increased numbers of infiltrating CD8+ cytotoxic T cells [17, 27], which is consistent with the

postulate that hypermutated gliomas are more immunogenic.

Hypermutated gliomas have been a subject of intense investigation for some time,

though the reported frequencies of hypermutation vary markedly due to differences in cohort

selection. In our screening of over 660 untreated sporadic grade II-IV TCGA gliomas in GlioVis

[5], only 15 had detectable mutations in DNA repair enzymes (not shown). But a study by the

TCGA consortium showed that 7/19 (36%) TMZ-treated GBMs were hypermutated [1]. Johnson

et al. reported hypermutation patterns in 6/10 (60%) post-TMZ tumors, and suggested that most

of the acquired mutations were likely directly induced by TMZ [14]. In a separate study of 114

matched pre- and post-treatment GBMs, 17 (15%) showed a hypermutator profile at recurrence;

among those 17 cases, 16 had mutations in MMR genes, and showed enrichment for MGMT

methylation and IDH1 mutation [26]. Others have verified the association between IDH1

mutations and hypermutation after TMZ [14, 25, 30]. Among 157 pediatric gliomas, only 9 (6%)

were hypermutated, and 7 contained mutations in DNA repair genes [13]. In our own cohort,

9/100 gliomas were hypermutated, all 9 had been previously treated with TMZ, all 9 had MGMT

promoter methylation, and 5/9 were IDH1 mutant (Table 2). Screening for hypermutation-

associated MMR defects therefore appears to be of greatest value in recurrent, post-TMZ

gliomas, especially MGMT-methylated and/or IDH1 mutant tumors (Table 3 and Table 4).

Although the Msh2, Msh6, Mlh1, and Pms2 IHC panel is used to screen colorectal

cancer, mutations in other DNA repair genes have also been reported in post-TMZ

hypermutated gliomas, including MSH4, MSH5, MLH3, PMS1, POLE, and POLD1 [5, 10, 13,

26]. In our own cohort, we found a hypermutated glioma with an inactivating mutation in yet



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another gene associated with DNA repair, ATM (Table 2) [3]. Thus, while the MMR IHC panel

designed for colorectal cancer detects most hypermutated gliomas, it may be helpful to add

more IHC markers for gliomas.

Interpretation of MMR IHC in gliomas is relatively straightforward, especially since

nonneoplastic cells within the glioma serve as a reliable positive control (Figure 1). MMR

staining is lost in areas of necrosis and thermal artifact (not shown), so such regions should not

be scored.

Thus far, results from immune checkpoint inhibitors in gliomas have been mixed [6, 15,

21, 29]. While at least partial responses to immune checkpoint inhibitors have been observed in

patients whose sporadic gliomas had elevated TMB [8, 16], the best responses have mostly

been in glioma patients with an inherited defect in an MMR gene, where 100% of the glioma

cells have MMR deficiency [4, 12, 23]. Our data showing frequent heterogeneity of MMR loss in

hypermutated gliomas (Figure 2 and Table 2) underscores the fact that TMB is just a

mathematical average of the specimen that was submitted for NGS, and that subclones may

exist in “hypermutated” gliomas that are not necessarily hypermutated. Conversely,

hypermutated subclones could potentially exist in tumors whose overall TMB has not yet

reached the widely accepted cutoff of 20 mutations/Mb, although we did not see this in our own

cohort (not shown).

In sum, DNA MMR enzyme IHC can serve as a rapid, low-cost method of screening for

hypermutated gliomas. Highest yield for screening includes recurrent gliomas with MGMT

promoter methylation and/or IDH1 mutations. While the current panel used for colorectal

cancers has very good sensitivity and excellent specificity, adding more DNA repair markers

would further enhance its value. Finally, it may be valuable to consider heterogeneity in

hypermutated gliomas as a possible predictor of immunotherapy response.



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CONFLICTS OF INTEREST

The authors have no conflicts of interest to disclose.

AUTHOR CONTRIBUTIONS

CH and MM designed the project, performed data collection, and scored all cases. KM, RJ, and

KK collected the cases, and AS performed MMR IHC. MM and CH wrote the manuscript.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant R01NS102669 (C.H.) and by the

Northwestern SPORE in Brain Cancer P50CA221747. MM was supported by an institutional

grant from the Northwestern University Department of Pathology Resident Research Committee.

The authors thank Kimberley Liggett and Juan Cuellar for their assistance with slide retrieval.



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age mean 49.6 years (range 19-85) variable N sex

male 46 female 54

WHO grade

I 4 II 11 III 15 IV 70

histotype

astrocytoma 91 oligodendroglioma 9

IDH1 status

wild-type 66 mutant 34

MGMT status

methylated 46 unmethylated 54

TMZ treatment

pre 69 post 31

TOTAL 100

Table 1: Cohort characteristics. Since cohort N=100, numbers are the same as percentages.

TMZ=temozolomide.



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Figure 1: MMR IHC in Case 1. The tumor was a recurrent GBM, post-TMZ therapy, in a 57

year-old woman (Table 2). Tumor cells showed loss of Msh2 (a) and Msh6 (b), and retention of

Mlh1 (c) and Pms2 (d). Note the normal immunostaining within nonneoplastic cells scattered

throughout the tumor in (a) and (b). Scale bar=100 µm.



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Table 2: Key characteristics of all identified hypermutated gliomas. TMB=tumor mutation burden, AF=allelic fraction,

IHC=immunohistochemistry.

case pre or post TMZ

IDH1 status TMB MGMT

methylation Msh2 Msh6 Mlh1 Pms2 MMR mutations (AF)

pattern of MMR IHC loss

1 post wt 67.8 positive lost lost retained retained MSH2 stop gain (33.0%) homogeneous

2 post wt >50.0 positive lost lost retained retained

MSH2 stop gain (14.8%)


homogeneous

3 post mut 108.7 positive retained lost retained retained


MSH6 frameshift (16.6%)

homogeneous

4 post mut 65.0 positive retained retained lost lost PMS2 missense

(7.2%) MLH1 copy loss

heterogeneous

5 post mut 70.5 positive lost lost retained retained MSH2 stop gain (74.0%) heterogeneous

6 post mut 27.0 positive lost lost retained retained

MSH2 splice variant (37.8%)


homogeneous

7 post wt 58.0 positive lost lost retained retained


MSH6 missense (29.4%)

heterogeneous

8 post mut 20.8 positive lost lost retained retained

PMS1 missense (32.5%)

MSH6 missense (11.2%)

heterogeneous

9 post wt 29.5 positive retained retained retained retained ATM splice variant (10.5%) none

All rights reserved. N

o reuse allowed w

ithout permission.

was not certified by peer review

) is the author/funder, who has granted m

edRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (w

hichthis version posted N

ovember 15, 2019.

; https://doi.org/10.1101/19012005

doi: m

edRxiv preprint

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16

age sex WHO

grade astrocytoma IDH1 mutant

MGMT methylated TMZ-treated hypermutant

age 1 sex 0.053 1

WHO grade 0.6 0.084 1 astrocytoma 0.28 0.01 0.48 1 IDH1 mutant -0.50 0.058 -0.36 -0.44 1

MGMT methylated -0.20 -0.006 -0.11 -0.27 0.48 1 TMZ-treated 0.15 0.075 0.39 0.21 -0.12 0.16 1 hypermutant 0.046 0.06 0.18 0.099 0.14 0.34 0.47 1

Table 3: Correlation matrix. TMZ=temozolomide.

All rights reserved. N

o reuse allowed w

ithout permission.

was not certified by peer review

) is the author/funder, who has granted m

edRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (w

hichthis version posted N

ovember 15, 2019.

; https://doi.org/10.1101/19012005

doi: m

edRxiv preprint

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17

variable regression coefficient

TMZ-treated 0.19 MGMT methylated 0.083

IDH1 mutant 0.024 astrocytoma 0.013

age 0.0037 sex 0.001

WHO grade -0.0004 Table 4: Multiple regression results. Variables are listed from largest to smallest regression

coefficients, relative to hypermutation. TMZ=temozolomide. Adjusted multiple R2 = 0.26 with

standard error=0.24.



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Figure 2: Heterogeneous MMR IHC in hypermutated gliomas. In Case 5, which was a post-

TMZ IDH1 mutant GBM in a 44 year-old woman (Table 2), clusters of tumor cells retained Msh2

and Msh6 positivity, but were surrounded by Msh2/6-deficient cells (a, b). Case 7 was a post-

TMZ IDH1 wild-type GBM in a 65 year-old woman (Table 2). Msh6 was lost in many glioma

cells (c), but under high power, it was apparent that a subset of cells with identical tumor nuclear

morphology retained Msh6 (d, red asterisk). Also note the smaller rounded nuclei in (d), which

are most likely lymphocytes and/or oligodendrocytes. Scale bar=100 µm in (a, b, c) and 25 µm

in (d).



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